Diabetes is one of the leading causes of death by disease worldwide. Type I diabetes, is a major form of the disease that typically develops at a young age and results from autoimmune destruction of islet beta-cells with consequent insulin deficiency and dependence on exogenous insulin treatment. Insulin therapy is the major intervention for the treatment of type I diabetes, however, insulin not a cure as it is not always possible to maintain blood glucose levels within a narrow physiological range using insulin and it does not prevent the progression of the disease and severe diabetic complications that eventually arise. Pancreatic islet transplantation is also an effective therapy (29) but is limited largely by the limited resources of human islets. In addition, immune-suppressors need to be used for life in the islets-transplanted patients.
Apoptosis is the main cause of the death of beta-cells in type I diabetes. Under normal conditions, maintenance of beta-cell mass is a dynamic process, undergoing both increases and decreases to maintain glucose levels within a narrow physiological range (1; 2). In subjects with obesity and insulin resistance associated diabetes, diabetes occurs only when the beta-cells lose their compensatory capacity. Diabetes does not occur even in the presence of insulin resistance if the beta-cell mass is maintained or enhanced. In type I diabetes, beta-cell apoptosis occurs as a result of autoimmune destruction involving T cell infiltration of the islets of Langerhans (5-7). The progressive destruction of the pancreatic beta-cells is largely due to lymphocytic infiltration of the islet, resulting in insulin deficiency. IL-1beta, TNF-alpha and IFN-gamma are released by macrophages and T cells during this autoimmune response and are important mediators of beta-cell destruction (5; 23; 24) via a mechanism that involves apoptosis and necrosis (24).
GLP-1 is a major physiological insulinotropic hormone which is secreted from the enteroendocrine L cells of the intestinal tract in response to nutrient ingestion (12-14). GLP-1 enhances pancreatic islet beta-cell neogenesis/proliferation and inhibits beta-cell apoptosis; in a glucose-dependent fashion (15; 16). GLP-1 also augments insulin secretion and lowers blood glucose in rodents as well as in humans in both type I diabetes (17; 18) and type II diabetes (19; 20). Recent studies have demonstrated that in insulin-secreting beta-cells, the apoptosis and necrosis induced by cytokines could be significantly blocked by glucagon-like peptide-1 (GLP-1) or exendin-4 (Ex4), a long-acting potent agonist of the GLP-1 receptor (24; 25). In vivo studies have shown that treatment with GLP-1/Ex4, stimulated beta-cell neogenesis in streptozotocin (STZ)-treated newborn rats resulting in persistently improved glucose homeostasis at an adult age (26). In type I diabetes patients, treatment with Ex4 normalized postcibal glycemic excursions (18). It is believed that the mechanism by which GLP-1 modulates beta-cell mass involves primarily 1) enhancement of β-cell proliferation, 2) inhibition of apoptosis of B-cells and 3) beta-cell neogenesis (13; 27; 28).
The GLP-1 receptor (GLP-1R) is a G-protein coupled receptor (GPCR) that is expressed mainly by pancreatic beta-cells and to some extent by cells of other tissues (lungs, heart, kidney, GI tract and brain), and is coupled to the cyclic AMP (CAMP) second messenger pathway (13; 21). Activation of other protein kinases including Akt (protein kinase B) (3; 13; 22) is found to be important in mediating GLP-1 action in promoting beta-cell growth and inhibiting apoptosis. In animals models of type II diabetes, it has been recently demonstrated that treatment of GLP-1 or exendin-4 (Ex4) prevented onset of diabetes (3; 4) by enhancing beta-cell growth and inhibiting apoptosis (3; 22). GLP-1 has many attractive biological actions, and demonstrated clinical efficacy in type II diabetes (9). Beta-cell replication and neogenesis are predominant mechanisms underlying beta-cell mass expansion. In addition, prevention of beta-cell apoptosis is important. GLP-1 has been found useful in the treatment of type II diabetes, which is consistent with its beneficial effects on beta-cell survival, function and growth. It has been demonstrated that expansion of beta-cell mass by treatment with glucagon-like peptide-1 (GLP-1) prevented the onset of diabetes in animal models predisposed to type II diabetes (3; 4). U.S. Pat. No. 6,899,883 and U.S. Pat. No. 6,989,148 disclose methods of treating type I diabetes using insulin and glucagon-like peptide 1(7-37) or glucagon-like peptide 1(7-36) amide.
The major obstacle in treating patients with native GLP-1 is its short circulating half-life (t1/2<2 min) that results mainly from rapid enzymatic inactivation by dipeptidyl-peptidase IV (DPP-IV) (30; 31), and/or renal clearance (32). Therefore, continuous subcutaneous infusion by pump is necessary to maintain GLP-1 action in vivo (33). Though DPPIV inhibitor can also increase the half-life of GLP-1 and are being tested in clinical trials. However, this approach lacks specificity, as DPPIV also inactivates several other peptide hormones and some chemokines (9), and its inhibition may lead to adverse reactions. In this respect, significant efforts have been made to develop pharmaceutical long-acting degradation-resistant GLP-1 mimetic peptides. Human GLP-1 analogues with amino acid substitutions (34-36) and/or N-terminal modifications including fatty acylated (37; 38) and N-acetylated (38) modifications exhibit significantly prolonged circulating t1/2, and potently reduce glycemic excursion in diabetic subjects (37). Ex4, a reptilian peptide with high sequence homology to mammalian GLP-1 is a potent GLP-1R agonist (39). Furthermore, albumin protein-conjugated GLP-1 also has the anti-diabetic and other beneficial activities of GLP-1 along with a prolonged half-life (40).
It is likely, however, that in some patients derivatives of GLP-1 will eventually be recognized and neutralized by humoral immunity, as observed with various peptides such as human growth hormone or insulin (41; 42) and, indeed, also Ex4 (39). This can occur either because the protein is foreign (i.e. Ex4), or because it is administered with a vehicle or by a route that promotes immunity. This is initiated when B lymphocytes that have a reactive immunoglobulin receptor (B-cell receptor [BCR]) bind to the hormone. However, there is evidence that B-cell stimulation can be prevented by co-ligating inhibitory receptors. B-cell stimulation is blocked when the BCR is cross-linked with FcγRIIB receptors that bear cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIM) (43-47). It is thought that B cell reactivity to GLP-1 will be prevented or diminished when this peptide is fused to an Fc segment, through binding of this Fc segment to the FcγRIIB receptor. This is consistent with the tolerogenic effects of IgG carrier proteins, as demonstrated extensively in many studies (48). A second important consideration is that peptide drugs can give rise to dangerous anaphylactic reactions. For instance, fatal anaphylaxis in nonobese diabetic (NOD) mice has been described after repeated subcutaneous insulin peptide B:9-23 immunizations (49). These anaphylactic reactions result from the production of IgE antibodies against the therapeutic peptide, resulting in classic type I hypersensitivity reactions. A recent study (50) suggests that this anaphylactic response can be blocked by fusing the allergen with IgG-Fc that bind to FCγRIIB of mast cells or basophils and prevents degranulation. Furthermore, administration of GLP-1/Ex4, combined with immunosuppression by polyclonal anti-T cell antibody, induced complete remission in 88% of overtly diabetic NOD mice (8). However, limitations of this strategy are 1) immunity to Ex4 which has >45% variation of amino acid sequence compared to native GLP-1 (9) and 2) systemic suppression of immunological responses by an anti-T-cell antibody that may lead to adverse immunologic effects (10). Because autoimmunity is persistent in type I diabetes subjects, control of autoimmunity with immunosuppression (preferably specifically directed against the autoaggressive T cells) is necessary for the replacement of islet cells and definitive treatment of this disease.
Gene therapy has been attempted in animal models of diabetes. For example, gene therapy has been directed at the systemic delivery of regulatory cytokines (e.g., IL-4, IL-10, TGF-beta-1) (54; 55), or at modification of islet cells ex vivo with some of these genes prior to transplantation (56). Alternatively, investigators have transfected islet cells ex vivo with genes such as bcl-2 that prevent apoptosis (56-58). The introduction of genes into transplanted islets has been limited by incomplete protection against anti-islet immunity, and the relatively short period of expression of some vectors (59). In addition, gene therapy to deliver insulin (or insulin analogues) in liver, muscle or other tissues has been accomplished, although physiological regulation of blood glucose levels has not been achieved and is a major limitation (62; 63). An alternative involves delivery of a gene(s) (e.g., PDX-1) in vivo to induce islet-cell differentiation of liver cells (64), but initial reports of success have been difficult to duplicate. Another important factor is that current proposed therapies fail to control autoimmunity effectively and as long as the autoimmune response of type I diabetic subjects is not controlled, new islets whether transplanted or produced by regeneration will be rejected. Indeed, many potential gene-based approaches have been proposed over the years, but none appears readily applicable to humans. These therapies have almost all been based on viral-vectored gene transfer which has limitations, particularly in terms of pathogenicity and immunogenicity and thus do not provide an effective and safe therapeutic method. For example, U.S. Pat. No. 6,991,792 discloses a method for delaying onset of type 1 diabetes mellitus using a vaccine comprising a recombinant vaccinia virus incorporated with a gene for coding glutamic acid decarboxylase.
Many forms of immunotherapy ameliorate diabetes in NOD mice (53), although most are effective only if initiated prior to the onset of the disease. Unfortunately, most patients initially present with diabetes. More recently, CD3 monoclonal antibody (mAb) therapy was found effective after the onset of disease in NOD mice, and acts by inducing regulatory T cells (Tr) (67). However, recent clinical trials suggest that CD3 mAb therapy by itself delays beta-cell loss, but cannot return patients to normoglycemia (10; 68). This is presumably because newly diabetic patients have a limited number of residual islet beta-cells, which are not sufficiently protected or replenished by this treatment. Another limitation is that most forms of immunotherapy (as in the case of CD3) are not specific to the autoaggressive T cells, and affect many other immune responses, possibly causing undesirable effects. Notably, administration of ChAglyCD3 (a humanized CD3 mAb) was frequently associated with a cytokine release syndrome and transient Epstein-Barr viral mononucleosis (10). The long-term effects and safety of this method needs to be further assessed.
Thus, there is a need to develop effective treatment strategies that target the molecular mechanisms underlying type I diabetes rather than its consequences.